Average distributions unwashed biopsies

**(b)** 

**Figure 5.** *Cont*.

**Figure 5.** (**a**) The average relative abundance of the ten most common bacterial genera of untreated biopsies from dyspepsia patients and gastric cancer patients. Comparison of (**b**) the alpha-diversity shown by a Shannon Index, *p* = 0.556831 and (**c**) the beta-diversity between the two groups shown by PCoA plot, *p* = 0.052947, in biopsies from dyspepsia patients (red) and gastric cancer patients (blue).

A low percentage of the bacterial reads in biopsies from dyspepsia patients were identified as *Lactobacillus* spp. (<0.1% of total bacterial reads). Most biopsy pairs from gastric cancer patients (30 pairs) did not show presence of *Lactobacillus* spp.; 6 biopsy pairs showed a relative abundance of 0–0.5% of total bacterial reads, and 7 biopsy pairs showed a relative abundance of 0.5–2% *Lactobacillus* spp., while three biopsy pairs showed a high relative abundance of *Lactobacillus* spp., which both increased (from 11 to 27%) and decreased (from 23% to 0.4% and from 29% to 5%) between the unwashed and washed biopsies. This difference in *Lactobacillus* spp. abundance was not significant between dyspepsia and gastric cancer patients.

The biopsies from antrum and cancer area of gastric cancer patients showed no significant differences in the distribution of bacterial groups (Figure 6a, Table 6).

The diversity of bacteria in biopsies from antrum and corpus did not show differences in the bacterial diversity within the sample areas (Figure 6b) or between the two sample areas (Figure 6c).


**Table 6.** Mean and standard error as percentage of the 10 most common bacteria as percentage of total bacterial reads in the microbiome analysis of biopsies from gastric cancer patients (*n* = 12).

**(a)** 

 **Figure 6.** *Cont*.

**Figure 6.** (**a**) The relative abundance of bacteria in untreated biopsies from the antrum and corpus area of gastric cancer patients. No significant differences were observed in the distributions of bacteria between the two groups. (Percentages are listed in Table 6). Comparison of (**b**) the alpha-diversity shown by a Shannon Index, *p* = 0.960995 and (**c**) the beta-diversity between shown by PCoA plot, *p* = 0.111888, in biopsies from antrum area (red) and corpus area (blue) in the stomach.

#### *3.3. Presence of* H. pylori

± ± *H. pylori* was detected in 14 of 34 patients (41%) by histology. Culture of *H. pylori* was only successful for 4 biopsies from 2 patients, despite incubation for additional days. Microbiome analysis identified DNA from *H. pylori* in 16 of 34 patients (47%), and *H. pylori* was identified as the only species of *Helicobacter* in the biopsies. The difference between culture and 16S rRNA gene amplicon sequencing may be explained by the fastidious nature of *H. pylori*, which may be difficult to culture after storage.

Three different distribution types of *H. pylori* and non-*Helicobacter* were observed; 4 biopsy pairs showed almost complete dominance of *H. pylori* (>90% of total bacterial reads in at least one of the biopsies) (Figure 7a), 16 biopsy pairs showed a mixed relative abundance of *H. pylori* and other bacteria (Figure 7b), and 26 biopsy pairs showed none or less than 1% of total bacterial reads identified as *H. pylori* (Figure 7c).

The biopsies determined positive or negative for *H. pylori* by histology showed significant differences in the bacterial diversity. The *H. pylori*-negative biopsies showed a significantly higher diversity than *H. pylori*-positive biopsies (*p*-value = 0.004353) (Figure 8a). In addition, a significant difference in the bacterial diversity was observed between biopsies determined positive or negative for *H. pylori* (*p*-value = 0.0009999), indicating that the presence of *H. pylori* may change the bacterial community to allow for a unique composition (Figure 8b).

Group 1 - Dominance of *H.pylori*
